KillerRed, a red fluorescent protein, is a photosensitizer that efficiently generates reactive oxygen species (ROS) when irradiated with green light. Because KillerRed is genetically encoded, it can be expressed in a spatially and temporally regulated manner under control of a chosen promoter and thus is a powerful tool for studying the downstream cellular effects of ROS. However, information is still limited about the effects of KillerRed-mediated production of ROS inside the mitochondria (mtROS). Therefore, we investigated whether mtROS generated by KillerRed could trigger mitochondrial damage and cell death by generating human cell lines (HEK293T and HeLa cells) that stably expressed mitochondria-targeting KillerRed (mtKillerRed). We found that mtROS generated by mtKillerRed caused depolarization of the mitochondrial membrane and morphological changes, which were partly due to the mitochondrial permeability transition (MPT), as well as inducing both caspase-dependent cell death (apoptosis) and caspase-independent cell death. In order to study the pathological processes initiated by mtROS in animals, transgenic Caenorhabditis elegans expressing mtKillerRed in muscle tissue were generated. Transgenic larvae showed developmental delay following light irradiation, suggesting that mtROS influenced the development of C. elegans larvae. In conclusion, our studies demonstrated that the photosensitizer KillerRed is effective at inducing oxidative damage in the mitochondria, and indicated that our experimental systems may be useful for studying the downstream cellular effects of mtROS.